In vitro translation system

In vitro methylation assays were performed as described previously (Bedford et al., 2000 (link)). In brief, GST-PRMT1 (mouse), PRMT3 (mouse), PRMT4 (mouse), PRMT6 (Rat), and various GST-Smurf2 constructs were purified using Glutathione-Agarose 4B (Peptron). Various GST-Smurf2 constructs (0.5 μg) were incubated with indicated GST-PRMTs (0.2–1.0 μg) in the presence of 2 μl of S-adenosyl-L-[methyl-³H] methionine ([³H]AdoMet; 83.3 ci/mmol, Perkin Elmer) for the indicated time at 30°C in 20 μl of methylation reaction buffer (15 mM Tris-HCl (pH.7.5), 25 mM NaCl 10 mM EDTA). Methylation reactions were stopped by the addition of 4X SDS sample buffer, followed by heating 100°C for 10 min. Samples were separated on SDS-PAGE and stained with 0.05% Coomassie Brilliant Blue. After destaining, gels were soaked in EN³HANCE (PerkinElmer) according to the manufacturer’s instructions and visualized by fluorography after exposure for 1 day to 1 week at −80°C.
For in vitro binding assay, bacterially expressed GST-Smurf2 were purified using glutathione-agarose beads (GE Healthcare) and incubated with [S³⁵]-labelled Myc-PRMT1, which was generated by an in vitro translation system (Invitrogen). After immunoprecipitation, the samples were analyzed by SDS-PAGE, followed by immunoblotting.

Cells were lysed in lysis buffer (20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1.0% Triton X-100, 20 mM NaF, 2 mM EDTA, 2 mM Na-orthovanadate, 1 mM PMSF, and 5 μg/ml leupeptin A), after which the concentrations of proteins were measured using Bradford reagent (Bio-Rad). For immunoprecipitation, 600–800 μg of cell lysates was incubated with proper antibody in lysis buffer overnight at 4°C with constant rotation. For in vitro binding assays, GST-fused proteins were expressed in BL21 bacterial cells and purified using glutathione-agarose beads (GE Healthcare). Proteins harboring [S³⁵]-labeled methionine (Met) were generated by an in vitro translation system (Invitrogen). The immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. Mouse monoclonal anti-PRMT1 antibody (clone 171, Sigma), mouse monoclonal anti-mono/dimethyl arginine antibody (7E6, AbCam), anti-Flag monoclonal antibody (M2, Sigma), and anti-Smurf2 (D8B8, Cell Signaling) were used. Peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Santa Cruz biotechnology) were used, and proteins were detected by using enhanced chemiluminescence reagent (ELPIS or Milipore).